The IFN-lambda (IFN-λ) 1, 2, 3 cytokine family, also called IL-29, IL-28A, and IL-28B, respectively, has recently been identified (Kotenko et al., 2003; Sheppard et al., 2003). IFN-lambdas (IFN-λs) are potent immune-modulatory and anti-viral cytokines, recently implicated in clearance of Hepatitis C virus in humans. IL-28A (also named IFN-λ2), IL-28B (IFN-λ3) and IL-29 (IFN-λ1) are type III interferons that are class II cytokine receptor ligands. IFN-λs are related to type I IFNs (IFN-Is) as well as the IL-10 family of cytokines and signal via a heterodimeric receptor, consisting of one chain unique for IFN-λ (IFN-λ R1 or IL-28Rα) and another chain (IL-10R2), which is shared with IL-10 related cytokines. IFN-λs possess antiviral, antitumor and various immune modulating functions and in many ways resemble the function of IFN-Is (Li et al., 2009). In contrast to the ubiquitous expression of the IFN-I receptor, the expression of the IFN-λ receptor is restricted to limited cell types including epithelial cells and plasmacytoid dendritic cells (pDCs) (Ank et al., 2008; Sommereyns et al., 2008). Exposure to viruses or analogues of nucleic acids such as poly IC or CpG-oligonucleotides (ODN), conditions known to trigger the production of IFN-Is, also induce IFN-λs and largely depend on similar signaling components (Ank et al., 2008; Osterlund et al., 2007; Onoguchi et al., 2007). IFN-λs play a role in toll-like receptor (TLR) induced protection against mucosal viral infections and recent reports link the IL-28B gene with an ability to clear and recover from Hepatitis C infection (Ank et al., 2008; Ge et al., 2009). It is thus of utmost importance to understand the cellular origin of IFN-λs and the regulation of its production.
Several cell types have been described to produce IFN-λ including monocyte derived dendritic cells (DCs) and plasmacytoid dendritic cells (pDCs), but the cellular origin of double-stranded (ds) nucleic acid-induced IFN-λ in vivo is still elusive (Coccia et al., 2004; Ank et al., 2008; Osterlund et al., 2005). Monocyte derived DCs are not CD8+ conventional DCs (CD8+ cDCs) or equivalents of CD8+ cDCs (eCD8+ cDCs) since eCD8+ cDCs involve Fms-related tyrosine kinase 3 ligand (Flt3)-ligand (FL), but not GM-CSF, for development. Monocyte derived DCs fully depend on GM-CSF for development, even though GM-CSF might be combined with other cytokines such as IL-4 or TNF-alpha (TNF-α). GM-CSF dependent DCs are not equivalents of steady state DCs because the lack of GM-CSF or the GM-CSF receptor has no influence on the presence of normal pDC or cDC subsets in lymphoid organs (Naik et al. 2008). If cells are generated in vitro with the combination of GM-CSF and FL, only GM-CSF DC develop, but not pDCs or eCD8+ cDCs (Gilliet et al. 2002).
Polyinosinic:polycytidylic acid (poly IC) is a mimic of viral double stranded (ds) RNA generated during viral infections and it is recognized by TRIF-dependent TLR3 or Cardif (also known as IPS-1, MAVS, VISA)-dependent Rig-like helicases (RLH) in vivo. It is commonly used as an immune stimulant and it is an excellent adjuvant for the induction of Th1 CD4 T cell responses in a DC-targeted vaccine model (Longhi et al., 2009).
Conventional dendritic cells (cDCs) are not only effective antigen presenting cells but are also known as an innate source of cytokines. Among the mouse cDCs, a subset defined by the expression of CD8αα homodimers (CD8+) was identified as the major producers of IL-12p70 in various organs including spleen, lymph nodes, thymus and liver (Reis e Sousa et al., 1997; Hochrein et al., 2001; Pillarisetty et al., 2004). Another functional feature of CD8+ cDCs is their capacity for cross-presentation (Shortman et al., 2009).
The CD8+ cDCs are clearly a functionally distinct DC subset. However, these functional attributes may not always correspond with CD8 expression. Thus, apart from the CD8 molecule, other combinations of surface markers can be used to identify CD8+ cDC or their functional equivalents that may lack CD8 expression (eCD8+). Among CD11c+ MHC Class II high cells, various combinations of high expression of CD205, CD103, Necl2, Clec9a, CD24 accompanied with negative or low expression of CD11b and CD172a can be used (Hochrein and O'Keeffe, 2008; Shortman et al., 2009).
DC subsets can be generated in vitro from bone marrow precursor cells in the presence of Flt3-ligand (FL), FLDC (Brasel et al., 2000). The FLDC cDCs lack expression of CD8 and CD4, but using markers described above, they can be divided into functionally distinct subsets that resemble the spleen cDCs. One FLDC subset has been identified as the eCD8+ since it depends on the same transcription factors for development as CD8+ cDC, expresses several characteristic surface markers, such as high expression of Clec9a, but low expression of CD11b and CD172a and shows a similar expression profile of TLRs. Functionally, the eCD8+ DCs demonstrate a similar TLR-ligand responsiveness, as well as high IL-12p70 production and efficient cross-presentation. Upon in vivo transfer and recovery in the spleen, eCD8+ DCs express CD8 on their surface (Naik et al., 2005).
Expression of the different nucleic acid sensing systems TLR3, TLR7, or TLR9 and the RLHs varies among DC subsets (Hochrein and O'Keeffe, 2008). The downstream functions after engagement of these receptors also differ among the different DCs. pDCs predominantly use TLR7 and TLR9 for nucleic acid sensing, resulting in the high production of IFN-I and IFN-λs. Among cDCs, CD8+ cDCs highly express TLR3 but lack expression of TLR7 (Edwards et al., 2003). Furthermore, it has been found by proteomics that CD8+ cDCs, in contrast to CD8-cDCs, hardly express the RLHs and as a consequence are unable to detect the single stranded (ss) RNA viruses Sendai or Influenza virus (Luber et al., 2010).
CD8 is not expressed on human DC, whereas CD4 is expressed by all DC subsets, and thus other markers have to be employed to define human DC subsets and to possibly align the mouse and human counterparts. A set of antibodies designated BDCA1-4 has been established and is used to differentiate between pDCs and subsets of cDCs (Dzionek et al., 2000). Human BDCA3 positive DCs have been proposed as the human eCD8+ DC since they, as the mouse eCD8+ DC, selectively express high levels of Clec9a and Necl2, but only low amounts of CD11b (Shortman et al., 2009). Genome wide transcriptional analysis substantiated a close relationship of murine CD8+ cDC with human BDCA3+ cDCs (Robbins et al., 2008). As with the mouse eCD8+ cDCs, the human BDCA3+ cDCs have been found in various organs including blood, spleen, lung, tonsils, lymph nodes, colon and liver. Functional correlation between these human and mouse DC subsets are scarce although the CD11blow cDC of human thymus correlated with the mouse thymic CD11blow DC with high IL-12p70 production (Vandenabeele et al., 2001; Hochrein et al., 2001).
Miyake at al., 2009, describes that poly IC activates NK cells via IPS-1 and TRIF dependent ways. Both pathways were involved in B16 tumor suppression via NK cells. CD8a+ cDCs were identified as source of type I IFN (IFN-alpha/beta), IL-6 and IL-12p40 and responsible for the NK cell activation as measured by IFN-gamma production by NK cells.
Schulz et al., 2005, describes that, dsRNA present in virally infected cells is recognized by dendritic cells via TLR3. That, poly IC activates CD8a+ cDCs (increase of surface markers such as CD40, CD86, CD80 and gene activation of TNF-alpha, IL-6 and IFN-alpha/beta but only IL-6 protein could be detected). It was shown that TLR3 was necessary for this activation and that activated CD8a+ cDCs induced stronger CTL induction via cross-presentation.
Diebold et al., 2009, describes that replicon plasmid induce dsRNA intermediates which are detected by CD8a+ cDCs in a TLR3 dependent way. In contrast the activation of CTL was independent of TLR3.
WO 2006/054177 describes that certain tumors express TLR3 and that these tumors might be treated with TLR3-agonists such as poly AU.
WO 2009/088401 describes that combinations of TLR ligands with one of them being a TLR3 agonist would induce increased (adaptive) immune responses especially antigen specific CD8 T-cell responses. The claims also include activation of dendritic cells with combinations of TLR3 agonists and other TLR agonists and claim enhanced CD8 T-cell responses including enhanced cytokines produced by the T-cells.
WO 2004/060319 describes that combinations of TLR agonists and TNF/R agonist increase the amount of an antigen specific immune response. These antigen specific responses were either from T-helper cells (CD4 T cells) or Killer T cells (CD8 T cells).
WO 94/28391 describes that ligands for FLT3 can be used for hematopoietic stem cell or other immune cell expansion. Different forms of Flt3-ligands are described.
WO 2008/131926 describes that M-CSF can be used independent of Flt3-ligands or GM-CSF to induce the generation of dendritic cells. In particular the production of pDCs was independent of FL and of cDCs independent of GM-CSF.
Ank et al., 2008, describes that many different cell types produce IFN-lambda to TLR ligands or viruses. It also analyses the IFN-lambda receptor expression and uses in vivo virus infection models. Local application (intra vaginal) of poly IC or CpG-ODN protected mice from lethal intra vaginal HSV-2 challenge. It describes also that cDCs, pDC, B-cells T-cells and macrophages from the spleen produced IFN-lambda mRNA in response to HSV-2.
Sheppard et al., 2003, describes the existence of the IFN-lambdas and that they are related to IFN-I and IL-10 family of cytokines. It shows mRNAs for IFN-lambdas (IL-28A, IL-28B, IL-29), IFN-alpha and IFN-beta of human PBMCs after poly IC treatment or EMCV infection. The mRNA of the 3 IFN-lambdas and IFN-alpha and IFN-beta were upregulated upon exposure to either poly IC or virus.
O'Keeffe et al., 2002, describes the increase of DC subsets in response to various growth factors including showing the increase of CD8a cDCs in response to flt3-ligand. IL-12p40 and IL-12p70 production in response to CpG was analyzed and CD8a+ cDCs and after FL to ProGP (fusion protein of FL and G-CSF) CD8aint cDCs were the major producers of IL-12p70.
However, none of the above cited documents and patent applications provides a clue about cells which are the source of IFN-lambda.
It is therefore an object of the present invention to provide the specific type of cDC, which is the major producer of ds nucleic acid-induced IFN-λ.